PIDD Orchestrates Translesion DNA Synthesis in Response to UV Irradiation

Cell Death and Differentiation (2011) 18, 1036–1045 & 2011 Macmillan Publishers Limited All rights reserved 1350-9047/11 www.nature.com/cdd PIDD orchestrates translesion DNA synthesis in response to UV irradiation

E Logette1, S Schuepbach-Mallepell2, MJ Eckert1, XH Leo1, B Jaccard1, C Manzl3, A Tardivel1, A Villunger3, M Quadroni4, O Gaide3 and J Tschopp*,1

PIDD has been implicated in survival and apoptotic pathways in response to DNA damage, and a role for PIDD was recently identified in non-homologous end-joining (NHEJ) repair induced by c-irradiation. Here, we present an interaction of PIDD with PCNA, first identified in a proteomics screen. PCNA has essential functions in DNA replication and repair following UV irradiation. Translesion synthesis (TLS) is a process that prevents UV irradiation-induced replication blockage and is characterized by PCNA monoubiquitination and interaction with the TLS polymerase eta (polg). Both of these processes are inhibited by p21. We report that PIDD modulates p21-PCNA dissociation, and promotes PCNA monoubiquitination and interaction with polg in response to UV irradiation. Furthermore, PIDD deficiency leads to a defect in TLS that is associated, both in vitro and in vivo, with cellular sensitization to UV-induced apoptosis. Thus, PIDD performs key functions upon UV irradiation, including TLS, NHEJ, NF-jB activation and cell death. Cell Death and Differentiation (2011) 18, 1036–1045; doi:10.1038/cdd.2011.19; published online 18 March 2011

DNA lesions can result from endogenous metabolic pro- because p21 obstructs the interaction of PCNA with DNA cesses as well as from exogenous DNA damaging agents. In replication and repair factors.12–15 In vitro studies have shown both cases, different cellular responses are engaged (cell that p21 also inhibits the loading of PCNA onto DNA,7 thereby cycle arrest, repair pathways or apoptosis) to maintain genetic compromising DNA repair.9 UV is one of the major exogenous integrity. PCNA (proliferating cell nuclear antigen) acts as a sources of DNA damage. The most common photoproducts DNA sliding clamp, which helps loading of replicative DNA induced by UV irradiation are CPDs (cyclobutane pyrimidine polymerases. PCNA is involved in several forms of DNA dimers). As a consequence, replicative DNA polymerases are repair, such as NER (nucleotide excision repair), BER (base halted at DNA lesions, resulting in replication arrest. To avoid excision repair) and MMR (mismatch repair), and also in other this, two principal pathways are engaged that both require aspects of DNA metabolism, such as replication, chromatin PCNA: NER, the main repair pathway by which bases assembly and cohesion.1 PCNA mediates these different damaged by UV irradiation are replaced using the high-fidelity functions through interactions with proteins specific to each polymerases d and e and TLS (translesion DNA synthesis), a process.2 Many of these PCNA interacting proteins (PIPs) lesion bypass process that has evolved to avoid excessive contain a so-called PIP-box.1,3 Competition between different fatal arrest of replication during S phase.16 TLS is a partners for the same binding site on PCNA is one of the fundamental mechanism for tolerating DNA damage that mechanisms that coordinate the functions of PCNA in DNA has escaped repair, carried out by specialized low stringency replication and repair. PCNA is loaded around the DNA by TLS polymerases (the Y-family and B-family, for review see the conserved chaperone-like clamp loader complex RFC ref. 17), which synthesize across a wide variety of DNA (replication factor C), consisting of five subunits (RFC1 to lesions. Particularly in response to UV irradiation, polymerase RFC5).4,5 The RFC subunits all contain a PIP-box and thereby eta (polZ) has the unique property of incorporating nucleotides physically interact with PCNA.6 Once PCNA is linked to the opposite CPDs with similar efficiency than undamaged DNA, the RFC complex is ejected from the clamp to allow DNA.18,19 In mammalian cells, the major modification of DNA polymerase access to the clamp.7 PCNA in response to UV is monoubiquitination at lysine-16420 The protein with the strongest affinity for PCNA is the PIP- mediated by the Rad6-Rad18 complex.21,22 This mono- box containing p21 (Cip1/Waf1),8 a potent cyclin-dependent ubiquitination alters the affinity of PCNA for different kinase (CDK) inhibitor involved in cell cycle regulation, except polymerases, such as increasing the affinity for polZ, that is in response to UV, where cell cycle arrest is independent of then loaded onto the DNA and performs TLS past the UV p21.9 P21 binds to PCNA and inhibits its activity,10,11 mainly lesions.23,24 Even though the effect of p21 on PCNA function

1Department of Biochemistry, University of Lausanne, Epalinges, Switzerland; 2Department of Dermatology-Immunology, Geneva Medical Center, rue Michel Servet 1, Geneva, Switzerland; 3Division of Developmental Immunlogy, Biocenter, Innsbruck Medical University, Innsbruck, Austria and 4Protein Analysis Facility, Center for Integrative Genomics, University of Lausanne, Epalinges, Switzerland *Corresponding author: J Tschopp, Department of Biochemistry, University of Lausanne, Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland. Tel: þ 41 21 692 5738; Fax: þ 41 21 692 5705; E-mail: [email protected] Keywords: PCNA; PIDD; TLS; UV Abbreviations: TLS, translesion synthesis; NHEJ, non-homologous end joining; PIDD, p53-induced protein with a death domain; CPDs, cyclobutane pyrimidine dimers Received 04.10.10; revised 27.1.11; accepted 28.1.11; Edited by B Zhivotovsky; published online 18.3.11 Regulation of TLS by PIDD E Logette et al 1037 is still controversial, several reports describe p21 as an In co-immunoprecipitation experiments with overexpressed inhibitor of PCNA monoubiquitination and thus TLS is induced PIDD, we could confirm that PIDD interacts with PCNA, RFC5 by UV irradiation.9,25-27 (Figure 1a) and RFC4 (Supplementary Figure S1a) as well as PIDD (p53-induced protein with a death domain) was with the corresponding endogenous proteins (Figure 1b, initially described as a p53-inducible gene.28 PIDD contains lane 2). This interaction was specific as for example, the DD- N-terminal Leucine Rich Repeats (LRR), two intermediate containing FADD protein showed no affinity for PIDD ZU5 domains and a C-terminal Death Domain (DD).29 PIDD (Supplementary Figure S1b) and knockdown of PCNA and can act as a molecular switch able to activate either a pro- RFC5 resulted in the disappearance of the respective proteins survival or a pro-death program in response to DNA damage in the PIDD complex (Figure 1b, lanes 3 and 4). This depending on its auto-proteolytic processing and the compo- knockdown experiment also shows that the strong interaction sition of proteins incorporated into the so-called PIDDo- of PIDD with PCNA was not dependent on the presence of some.30 By recruiting RIP-1 and Nemo, PIDD activates the RFC5, and neither was the PIDD-RFC5 interaction dependent pro-survival NF-kB pathway,31 whereas recruitment of RAIDD on PCNA. The reconstitution of the PIDD-PCNA-RFC5 and caspase-2 to the complex sensitizes cells to apoptosis.29 complex by overexpression appeared to be independent of Recently, PIDD association with DNA-PKcs and caspase-2 the DNA damage response, as UV-C treatment did not alter was also shown to act on the NHEJ (non-homologous end- the composition of the complex (Figure 1a) (however, this UV joining) repair pathway in response to g-irradiation,32 suggest- independency observed with overexpressed proteins is in ing a role for PIDD and caspase-2 in DNA repair. Here, we contrast with the results obtained with endogenous proteins, identify PIDD as a new interaction partner of PCNA. We see below). provide evidence that PIDD modulates the p21-PCNA PIDD is constitutively auto-processed giving rise to a interaction and find that in response to UV-C, PIDD deficiency 48-kDa N-terminal fragment containing the LRR (PIDD-N) leads to a defect in PCNA monoubiquitination and a decrease and a 51-kDa C-terminal fragment (PIDD-C) containing the of polZ recruitment, whereas overexpression of PIDD has the death domain (DD). The latter undergoes further cleavage opposite effects. resulting in a 37-kDa fragment (PIDD-CC) (Figure 1d). Whereas initially formed PIDD-C mediates the activation of NF-kB, subsequent formation of PIDD-CC causes caspase-2 30 Results activation. PIDD, through its DD, was previously shown to interact with 29,31,32 PIDD forms a complex with PCNA and RFCs. To identify RAIDD, RIP1 and DNA-PKcs. We therefore investi- proteins that physically interact with PIDD, we gated whether this domain was also implicated in the immunoprecipitated Flag-tagged PIDD from stably interaction with PCNA. Using different non-cleavable mutants 30 transfected 293T cells, and analyzed PIDD-associated of PIDD previously described, we found that only the full- proteins by proteomics (Table 1). A substantial number of length protein and the PIDD-C fragment interacted with PCNA proteins were found that are known to have a role in DNA (Figure 1c). Taken together, these results indicate that PIDD replication and/or repair. Most notably, almost all members of forms a complex with PCNA and RFC5, which does not rely on the ‘PCNA-RFC-pold‘ complex were detected. We, thus, the DD and therefore is distinct from the caspase-2-activating decided to investigate a possible role of PIDD in DNA PIDDosome. More in-depth mapping experiments indicated replication and repair. that the Zu5 domains of PIDD are required for interaction with PCNA and RFC5 (Figure 1e). Deletion mutant of PCNA indicate that the C-terminal part of PCNA is important for Table 1 Functional categorization of PIDD-associated proteins identified by interaction with PIDD and especially the Zu5 domains proteomics analysis as described in supplemental experimental procedures (Figure 1f).

Category Representative proteins PIDD-PCNA interaction occurs transiently in the nucleus PIDD RAIDD/caspase-2 and is UV-C dependent. After a 1 h treatment of cells with Heat shock/chaperones — 100 J/m2 UV-C, there appeared to be no change in complex Replication factor PCNA/RFC5/POLd/TEL2 formation of overexpressed PIDD and PCNA protein (as homolog/MCM7 RNA polymerase RPB2/RPAP3 shown above in Figure 1a and Supplementary Figure S2a). Elongator complex PP6 As this was a surprising and unexpected observation DNA damage/repair DDB1/FANCI/DNA-PKcs/ (probably because of the overexpression system used), we XPD/XPG Histone modiﬁcation ANP32B investigated a possible effect of UV-C treatment on PIDD- Apoptosis HAX1/AIF PCNA interaction in a more physiological setting and found Cell cycle — that PIDD interaction with endogenous PCNA was indeed Ubiquitination TRIM32 2 Sumoylation SENP3 UV-C dependent (50 J/m ). A transient increase in the Nuclear organization/ NUP93 interaction occurring 30 min after irradiation was observed nucleoporins in the nucleus (Figure 2a). Most interestingly, the kinetics of Regulation of REDOX state PRDX1 the nuclear complex formation is in agreement with previous Transcription — results showing a peak of PCNA-based repair complex 33,34 Only proteins found in all experiments are listed. Data are representative of formation 30 min after UV irradiation. It is worth noting three independent experiments that the PIDD-CC fragment is absent in the nuclear

Cell Death and Differentiation Regulation of TLS by PIDD E Logette et al 1038

siRNA scr VSV- mock RFC5 PCNA PCNARFC5 Flag-PIDD - + + + Flag-PIDD - + +-++-++ Flag IP WB: PCNA Flag IP UV-C100 --+--+--+ WB: VSV WB: RFC5 * WB: Flag FL WB: Flag FL C C Lysate CC Lysate CC

WB: Flag WB: PCNA

WB: RFC5 WB: VSV WB: Flag C Lane : 1 2 3456789 CC Lane : 1234

Flag-PIDD Flag-IP - WT FL C CC WB: PCNA ID LRR WB: Flag FL 124 285 320 420 455 554 778 873 1 ZU5 ZU5 DD 910 PIDD-FL C S446 CC 446 ZU5 DD 910 PIDD-C Lysate S588 WB: PCNA 588 DD 910 PIDD-CC

WB: Flag FL 1 ZU5 445 PIDD-N

0) VSV-PCNA - FL ΔN ΔC Flag-PIDD-FL + - +-+-+- Flag-PIDD-Zu5 -++ -+-+- Zu5 (555-910) Δ Flag-PIDD mock FL (1-910)LRR (1-285)N (1-450)Zu5-Zu5C (300-60 Flag IP WB: VSV PCNA Flag IP WB: PCNA N-ter WB: RFC5 C-ter WB: Flag FL WB: Flag C

CC Zu5-Zu5 Lysate

Lysate WB: VSV PCNA WB: PCNA

WB: RFC5 N-ter C-ter

WB: Flag WB: Flag FL C

CC Zu5-Zu5

Figure 1 PIDD interacts with PCNA and RFC5 through its N-terminus. (a) PIDD interacts with overexpressed PCNA and RFC5: different VSV-tagged constructs were cotransfected in 293T cells with Flag-tagged PIDD and tested for interaction by co-immunoprecipitation (after 100 J/m2 UV-C irradiation where indicated). See also Figures S1a and S1b. (b) PIDD interacts with endogenous PCNA and RFC5: scramble or speciﬁc siRNAs were cotransfected with Flag-tagged PIDD or a control vector in 293T cells and interactions with endogenous RFC5 and PCNA were analysed by anti-Flag IP (* ¼ a long exposure was required to detect endogenous RFC5). (c) PIDD-CC fragment does not interact with PCNA: 293T cells were transfected with wt or different non-cleavable mutants of PIDD, and endogenous PCNA interactions were analyzed by anti-Flag IP. (d) Structure and processing of PIDD: amino acids positions of each domain are indicated (LRR, Leucine Rich Repeat; ID, Intermediate Domain; DD, Death Domain). (e) Different fragments of PIDD were overexpressed in 293T cells and interactions with endogenous RFC5 and PCNA were analysed by anti-Flag IP. (f) Deletion constructs of PCNA lacking or the C-ter (DC ¼ PCNA aa1–120) or the Nter regions (DN ¼ PCNA aa130–261), were cotransfected with Flag-tagged PIDD-FL or Zu5-Zu5 in 293T cells and tested for interaction by co-immunoprecipitation. (a–f) All the experiments were repeated at least two times. FL, PIDD Full-length; C, PIDD-C fragment; CC, PIDD-CC fragment as described in panel d (this nomenclature is conserved in all the ﬁgures)

immunoprecipitation, the major forms observed being the PCNA and PIDD-CC. The results suggest that the full-length full-length protein and the C-fragment, conﬁrming the and/or the PIDD-C fragment are important for the PCNA mapping experiments that showed no interaction between interaction. Similar kinetics and localization of PIDD-PCNA

Cell Death and Differentiation Regulation of TLS by PIDD E Logette et al 1039

cytoplasm nucleus Flag-PIDD --++++ + --++++ + UV-C Flag IP 50 -1h- 15’30’1h 2h - 1h - 15’30’1h 2h WB: PCNA

WB: Flag FL C CC Lysate WB: Flag FL C CC WB: PCNA

WB: p21

WB: Casp3

WB: Lamin Lane : 1234567 891011121314

cytoplasm nucleus cytoplasm nucleus UV-C [-]-15’30’1h2h [-] - 15’ 30’ 1h 2h PIDD IP 50 PIDD IP UV-C50 - 20’40’ 1h 2h -20’ 40’ 1h 2h WB: PCNA WB: PCNA IgG(H) WB: PIDD C C WB: PIDD CC CC Lysate Lysate WB: PCNA WB: PCNA WB: PIDD FL* WB: PIDD C FL CC C CC WB: p21 WB: p21 WB: Casp3 WB: Casp3 WB: Lamin WB: Lamin Lane : 1 2 3 4 5 6 7 8 9 101112 Figure 2 PIDD-PCNA interaction is regulated in nucleus by UV-C irradiation. (a) PIDD-PCNA interaction increases in nucleus after UV-C irradiation: 293T cells were transfected with Flag-tagged PIDD, subjected to cytoplasm-nuclear fractionation as described in ‘experimental procedures’, and endogenous PCNA interaction was analyzed in both fractions by anti-Flag IP after different times of 50 J/m2 UV-C irradiation. (b) Endogenous PIDD and PCNA interact in nucleus in response to UV-C irradiation: the same experiment as in figure (a) except that endogenous PIDD was immunoprecipitated. See also Supplementary Figure S2b. (c) Endogenous PIDD and PCNA interact in nucleus in a keratinocytes cell line: the same experiment as in figure (b) was performed in HaCaT cells. ([] ¼ IP control was done with a control IgG antibody, * ¼ a long exposure was required to detect PIDD-FL). (b and c) The hardly detectable endogenous PIDD-FL is only detectable with really high exposure (FL*). (a–c) The p21 degradation was analysed in parallel in total extracts. Caspase-3 and lamin were used as control of cytoplasmic and nuclear extracts, respectively. Data are representative of at least three repetitions interaction were also found when exclusively endogenous detaches from PCNA after UV-C treatment (Supplementary proteins were analyzed in 293T cells, in both total extracts Figure S2A, lane 6 versus 4) allowing PCNA to interact with (Supplementary Figure S2B) and nuclear fractions the other partners involved in DNA repair.11 The results (Figure 2b). To strengthen these data, we also tested the described above hinted at a possible role for PIDD in the formation of the complex in the HaCaT keratinocyte cell line process of p21 release from PCNA, as the peak of interaction (Figure 2c). Again, we observed a peak of interaction in the between PIDD and PCNA was at 30 min post-UV-C closely nucleus after 30 min UV-C irradiation, whereas no interaction coinciding with the start of p21 degradation (Figure 2a, was found between endogenous PIDD and PCNA in the lane 12). We therefore analyzed PCNA-p21 interaction, p21 cytoplasm. This probably means that the results shown in degradation and PIDD upregulation in more detail, and Figure 2a, where constitutive interaction was found in the found that all three events occur B30 min after UV-C cytoplasm were most likely because of overexpression of treatment (Figure 3a, lanes 4–6). To mimic upregulation of PIDD (in accord with Figures 1b and d). It is to note that the PIDD expression seen upon UV-C exposure, we expressed full-length form is well detected only using overexpression increasing amounts of Flag-PIDD, which consequently led system (Figure 2a), as the endogenous one is really hard to to PIDD binding correlating with p21 detachment from detect. However, and importantly, only in the nucleus of this PCNA in a dose-dependent manner (Figure 3b). This keratinocyte cell line, we could clearly detect the appearance suggests that at high-expression levels, PIDD is able to and upregulation of the endogenous full-length PIDD form in compete with p21 for PCNA binding. However, it is important response to UV-C irradiation (Figure 2c). to note that in this overexpression experiment, the levels of PIDD are probably higher than what would be found under PIDD overexpression induces dissociation of p21 from physiological conditions. In addition, p21 is highly expressed PCNA. The interaction between PCNA and p21 is modulated in most cells and is known to have a strong affinity for during the UV-C response (see Introduction section): p21 PCNA.12 Therefore, it is possible that under physiological

Cell Death and Differentiation Regulation of TLS by PIDD E Logette et al 1040

VSV-PCNA

VSV IP UV-C50 --10’ 20’ 30’ 45’ 60’ WB: p21

WB: PCNA VSV-PCNA -+++++ Lysate PIDD-Flag --0.1 0.5 1 2 WB: PCNA VSV- PCNA VSV IP WB: p21 PCNA WB: p21 WB: VSV

WB: Flag WB: PIDD C FL

CC C Lane : 1 2 3 4567 CC Lysate WB: VSV

VSV-PCNA WT D250-261 D2-9 WB: p21 Flag-PIDD -+-+-+ WB: Flag Flag IP FL WB: VSV C FL WB: Flag CC C VSV- PCNA CC Lysate Flag-PIDD -+-+ UV-C --+ + WB: VSV Flag IP 100 WB: VSV WB: Flag FL WB: Flag FL C C CC CC WB: p21 VSV IP WB: p21

VSV-PCNA WB: VSV sh mock PIDD UV -+ -+ Lysate VSV IP 50 WB: Flag C WB: p21 CC WB: VSV WB: VSV

Lysate VSV- PCNA WB: p21 WB: PCNA PCNA Lane : 1 2 3 4 WB: p21

WB: PIDD C

CC Figure 3 PIDD regulates the PCNA-p21 interaction in response to UV-C irradiation. (a–e) Where indicated, endogenous p21 interaction with PCNA was analysed by anti- VSV IP, endogenous PCNA interaction with Flag-tagged PIDD was analysed after anti-Flag IP, and p21 degradation and PIDD expression were checked in total extracts. (a) PIDD upregulation correlates with PCNA-p21 detachment in the course of UV-C: 293T cells were transfected with VSV-tagged PCNA and subjected to different times of 50 J/m2 UV-C irradiation. (b) PIDD overexpression is able to induce p21 detachment from PCNA: 293T cells were cotransfected with VSV-tagged PCNA and increasing amounts of Flag-tagged PIDD (the number indicates the ratio of Flag-PIDD plasmid transfected compared with the VSV-PCNA plasmid). (c) PCNA trimerization is not required for interaction with PIDD: 293T cells were cotransfected with differents constructs of VSV-tagged PCNA and Flag-tagged PIDD where indicated. See also Supplementary Figures S3a–c. (d) PIDD overexpression reduces p21-PCNA interaction in response to UV-C irradiation: 293T cells were transfected with VSV-tagged PCNA and with Flag- tagged PIDD and subjected to 100 J/m2 UV-C irradiation for 1 h where indicated. (e) PIDD deﬁciency leads to sustained p21-PCNA interaction in response to UV-C irradiation: 293T cells stably knocked-down for PIDD or the corresponding control cells, were transfected with VSV-tagged PCNA and subjected to 50 J/m2 UV-C irradiation for 30 min where indicated. All the ﬁgures are representative of at least three repetitions

conditions, PIDD upregulation ampliﬁes rather than induces interaction with PIDD. Another PCNA mutant (the KIE/A dissociation of p21 from PCNA upon UV-C irradiation. The substitution mutant),12 capable of forming trimers, but unable dissociation of p21 from PCNA would suggest that PIDD and to bind p21 (Supplementary Figure S3B), still interacts with p21 interact with PCNA in close proximity and compete for PIDD (Supplementary Figure S3C), indicating that the p21 nearby binding sites. We therefore used previously described and PIDD binding sites are distinct. In addition, this PCNA deletion mutants (D2–9 and D250–26112) that are competition is not because of direct binding of PIDD to p21 unable to form the trimeric PCNA structure and thereby as no interaction was found between these two proteins prevent p21 binding (Supplementary Figure S3A). PIDD was (Supplementary Figure S2A). Hence, it is highly likely that still able to interact with these mutants (Figure 3c) indicating the two binding partners of PCNA compete because of that the trimeric form of PCNA is not required for the steric hindrance.

Cell Death and Differentiation Regulation of TLS by PIDD E Logette et al 1041

sh mock PIDD si scr PIDD UV-C50 (h) 03 603 6 UV-C50(h) 0 1.5 3 6 0 1.5 3 6 WB: PCNA PCNA-Ub WB: PCNA PCNA-Ub PCNA PCNA WB: PIDD C WB: p-Chk1 CC WB: PIDD C WB: p-Chk1 CC

sh mock PIDD mock Flag-PIDD 24h HU (mM) 02.5 5052.5 UV-C (h) 50 0 0.5 1 3 6 0 0.5 1 3 6 WB: PCNA PCNA-Ub PCNA-Ub WB: PCNA PCNA PCNA WB: pChk1 WB: Flag C WB: PIDD C CC CC

PIDD +/+ -/-

mPIDD IP UV-C50 [-] - 15’ 30’ 1h 2h - mPIDD +/+ +/- -/- WB: PCNA UV-C50 (h) 062430624306243 WB:mPIDD C WB: PCNA PCNA-Ub CC PCNA Lysate WB:mPIDD C

CC +/+ +/- -/- WB:mPIDD C PCNA-Ub WB: PCNA CC PCNA Lane : 1234567 Figure 4 PIDD is involved in the monoubiquitination of PCNA in response to UV-C irradiation. (a–f) Cells were subjected to different times of 50 J/m2 UV-C irradiation or 24 h to different doses of HU and monoubiquitination of PCNA was analyzed by western blotting as described in ‘experimental procedures’. See also Figures S4b-c. (a) Hacat cells transfected with the indicated siRNAs (scr ¼ scramble) or without siRNA as control. (b) HeLa cells stably knocked-down for PIDD or corresponding control cells. (c) The 293T cells stably overexpressing Flag-PIDD or corresponding control cells. (d)asin(b). (e) Primary keratinocytes from PIDD þ / þ mice were subjected to different times of 50 J/m2 UV-C irradiation. Endogenous mPIDD was immunoprecipitated and endogenous PCNA interaction was analyzed ([] ¼ IP control was done with a control IgG antibody. Lane 7: keratinocytes from PIDD/ mice were also used as control). (f) Primary keratinocytes from PIDD þ / þ , PIDD þ / or PIDD/ mice (bottom panel show expression of mPIDD for each phenotype). All the ﬁgures are representative of at least two repetitions

As PIDD overexpression induces PCNA-p21 detachment methanesulfonate (MMS), mitomycine C, cisplatin and (Figures 3b and d, lane 2 versus 1), which is even more hydroxyurea (HU).23,25 PIDD deficiency also impaired PCNA pronounced in response to UV-C (Figure 3d, lane 4 versus 3), monoubiquitination in response to these different agents, we tested the PCNA-p21 interaction after UV-C irradiation in independently of the various cell lines used (Figure 4D, the absence of PIDD. In contrast to the overexpression Supplementary Figures S4A and S4B). experiments, we observed a sustained interaction between PIDD interacts with RIP1 and with RAIDD/caspase-2 in a PCNA and p21 after short exposures to UV-C in cells where DD-dependent manner, leading to the activation of NF-kB and PIDD had been knocked down by shRNA (Figure 3e), caspase-2, respectively. To determine whether these estab- confirming that PIDD has an active role in the destabilization lished signaling pathways are implicated in PCNA mono- of the PCNA-p21 interaction in response to UV-C. ubiquitination, we tested UV irradiation in cell lines knocked down for RAIDD and caspase-2, or overexpressing a PCNA-monoubiquitination in response to UV-C is PIDD- dominant negative form of IkBa, inhibiting the NFkB pathway. dependent. In response to UV-C, PCNA is monoubiquitinated In none of these cells was UV-induced monoubiquitination of concomitant with p21 dissociation and degradation.9 As PCNA affected (Supplementary Figures S4C, S4D and S4E), PIDD seems to act on the dissociation of p21 from PCNA, indicating that neither the NFkB pathway, nor the caspase-2- we investigated whether PIDD could promote PCNA PIDDosome activity was implicated. This is in agreement with monoubiquitination. Using both shRNA and siRNA strategies our previous experiments indicating that the PIDD-PCNA to reduce PIDD expression, monoubiquitination of PCNA in complex does not rely on the PIDD-CC fragment. We took UV-irradiated cells was indeed strongly reduced (Figures 4a advantage of the PIDD/ mice to confirm the results obtained and b), while overexpression of PIDD enhanced PCNA with cell lines in which PIDD expression was impaired with monoubiquitination (Figure 4c), confirming the important role siRNA. Cultures of mouse primary keratinocytes were of PIDD in this modification of PCNA. In contrast, generated and exposed to UV-C. We first confirmed the phosphorylation and thus activation of Chk1, a checkpoint interaction between endogenous PIDD and PCNA in kinase activated during the UV response, was not affected response to UV-C irradiation in these primary cells (Figure 4a). PCNA is also monoubiquitinated in response to a (Figure 4e), and observed, in agreement with experiments variety of other DNA damaging agents, including methyl obtained with cell lines, that keratinocytes of PIDD/ mice

Cell Death and Differentiation Regulation of TLS by PIDD E Logette et al 1042

sh mock PIDD Flag mock PIDD Discussion UV-C50 030’ 60’ 030’ 60’ UV-C50 030’ 60’ 030’ 60’ WB: PIDD C WB: Flag C DNA lesions that are not repaired before S phase are CC CC dangerous obstacles for the replication machinery, as most WB: PCNA PCNA-Ub WB: PCNA PCNA-Ub PCNA PCNA lesions cannot be accommodated into the active sites of the η WB: polη WB: pol replicative DNA polymerases, thereby blocking progression of the replication fork. Prolonged stalling of replication forks can Figure 5 PIDD regulates the localization of polZ to the chromatin in response to UV-C irradiation. (a and b) Cells were subjected to different times of 50 J/m2 UV-C then lead to a collapse of the replication machinery, possibly irradiation and chromatin extraction was performed as described in ‘experimental resulting in double-strand breaks and gross chromosomal procedures’. Relocalization of PIDD, PCNA and polZ to the chromatin was analyzed rearrangements, or even a permanent cell-cycle arrest with by western blot. (a) The 293T cells stably knocked-down for PIDD or corresponding consequential cell death. control cells. (b) The 293T cells stably overexpressing Flag-PIDD or corresponding To steer clear of this potential crisis, cells have evolved the control cells. These two experiments were done two times and showed the TLS bypass mechanism that handles stalled replication forks. same result One important feature of TLS is the switch from high fidelity replication polymerases to translesion polymerases that can showed a defect in PCNA monoubiquitination (Figure 4f). The incorporate either correct or incorrect nucleotides opposite the assembly of the PIDD-PCNA complex and the ubiquitination lesions demonstrating the disadvantage of the error prone of PCNA both occurred later in these primary cells as TLS mechanism. Yet TLS probably exists to avoid even worse compared with cell lines used before. damage caused by stalled replication forks. Bypass replication in response to DNA damage is directly controlled through covalent modifications of PCNA by mono- PIDD is required for polg localization to the chromatin in ubiquitin at residue Lys 164. Our results show that at least one response to UV-C. Monoubiquitination of PCNA, which is pathway leading to PCNA monoubiquitination and thus TLS the major modification of PCNA in response to UV-C, is requires the presence of PIDD. How PIDD influences PCNA associated with the recruitment of the polymerase polZ to the ubiquitination, however, remains speculative. One possible chromatin to act on TLS.23 As PIDD affects PCNA scenario is that upon PIDD upregulation induced by UV-C, monoubiquitination, we performed chromatin extraction on PIDD interacts strongly with PCNA thereby facilitates p21 293T cells after UV-C treatment and observed PCNA detachment from PCNA. This may support the loading of monoubiquitination, polZ recruitment as well as PIDD PCNA to the DNA (probably with the RFC complex), its recruitment to the chromatin, as expected (Figure 5a). Both monoubiquitination by the RAD6-RAD18 complex and inter- PCNA monoubiquitination and polZ recruitment were delayed action with polZ allowing TLS to be carried out. in PIDD-deficient cells (Figure 5a), whereas both events P21 is well known as an important effector of cell cycle were increased in response to UV-C when PIDD was arrest after various genotoxic insults. P21 was also shown to overexpressed (Figure 5b). modulate the monoubiquitination of PCNA and the interaction of PCNA with the TLS-specific polymerase Z.27 We found that PIDD strongly binds to PCNA thereby impacting on the PCNA- PIDD deficiency is associated with increased apoptosis p21 interaction. Sustained binding of p21 to PCNA after UV-C in response to UV-C. Efficient TLS is a crucial process in treatment in the absence of PIDD correlates with a decrease the prevention of cell death associated with stalled replication of PCNA monoubiquitination and polZ recruitment to the forks. Defects in TLS and polZ recruitment, as well as chromatin, in line with the inhibitory function of p21 in TLS. In prevention of PCNA monoubiquitination are associated with keeping with this, inhibition of p21 degradation by the sensitization to apoptosis induced by UV-C irradiation.27,35 proteasome inhibitor MG132 completely inhibited PCNA We thus investigated whether a defect in PCNA monoubiquitination correlating with a sustained PCNA-p21 monoubiquitination and polZ recruitment caused by PIDD interaction (Supplementary Figures S6A and B), which deficiency sensitizes cells to UV-C induced apoptosis. confirms that dissociation and degradation of p21 is neces- Figure 6a shows that HaCaT cells with transiently knocked- sary for PCNA monoubiquitination.9,25,36 down PIDD were more sensitive to apoptosis induced by PIDD is a multifunctional protein, which is constitutively UV-C (Figure 6). The same increase in cell death was autoprocessed. Although the PIDD-C fragment triggers the observed in HeLa cells in which PIDD expression was RIP1-mediated NF-kB pathway, PIDD-CC sensitizes cells to reduced by shRNA (Supplementary Figure S5A). apoptosis through recruitment of RAIDD/caspase-2.30 A third To reproduce these results in a more physiological setting, function is mediated by the PIDD-DNA-PKcs-caspase-2 we irradiated mice with UV-B to test the consequences of an complex that is implicated in the maintenance of a G2/M impaired TLS in vivo. In agreement with our previous in vitro DNA checkpoint and DNA repair by the NHEJ pathway.32 The experiments, epidermis from irradiated PIDD/ mice showed involvement of PIDD in TLS is a fourth function that appears to higher sensitivity to cell death induced by UV-B irradiation act independently of the three other complexes. Neither compared with WT as indicated by the higher number of cells caspase-2, RAIDD deficiency nor NFkB inhibition influences containing active caspase-3 (Figures 6c and d and Supple- PIDD-induced PCNA monoubiquitination. This ‘indepen- mentary figure S5B). The results indicate that PIDD deficiency dence’ is in agreement with the fact that, in contrast to the participates in the sensitization of cells to apoptosis induced DD-dependent interaction with RIP1, RAIDD and DNA-PKcs, by UV irradiation in vivo. it is the N-terminal part and most probably the Zu5 domains of

Cell Death and Differentiation Regulation of TLS by PIDD E Logette et al 1043

si scr PIDD UV-C (h) Lysate 50 031.5 60 1.5 3 6 siRNA proform 100 WB: C3 scr cleaved 80 PIDD fragments 60 WB: PIDD C 40 CC

Apoptosis (%) 20 WB: Bid proform 0 tBid* UV-C (h) 069 Digitonin Extracts 50 WB: cyt c

P = 0.0125 Pidd +/+ Pidd -/- * 200

160 HE staining 120

+/+ -/- Pidd Pidd 80

40 cells per 20 random fields) Active Caspase-3 (number of 0 Caspase--3 staining Pidd -/- Pidd +/+ UV-B 200mJ/cm2 Figure 6 PIDD deficiency sensitizes cells to UV-C induced apoptosis. (a and b) Hacat cells were transfected with the indicated siRNA and subjected to different times of 50 J/m2 UV-C irradiation. (a) Markers of apoptosis, Bid and caspase-3 cleavage as well as Cytochrome c release were analysed by western-blotting. See also Supplementary Figure S5A. (b) Apoptosis was quantified by Hoechst staining and show the mean of two independent experiments. (c and d) PIDD þ / þ or / mice were irradiated as described in ‘experimental procedures’. Apoptosis was detected by HE staining (arrowheads indicate sunburn cells) and active caspase-3 staining (c) and quantification (d). See also Supplementary Figure S5b. Data shown are representative of at least three independent experiments and reported as mean ±S.D. P-value for statistical significance was calculated using the Student’s t-test and considered significant (*)

PIDD that are responsible for its interaction with PCNA and PIDD-DNA-PKcs-caspase-2 complexes are unlikely to be RFC proteins. It is currently not clear if PIDD-FL, the PIDD-C implicated. Yet, it has recently been found that caspase-2 form or both are responsible for the interaction, in part activation can also occur independently of the PIDDosome.37 because the endogenous full-length form is hardly detectable It remains to be shown therefore whether caspase-2 is by western blotting. implicated in UV-induced cell death. At least in vivo, skin of We identiﬁed in PIDD a sequence that could resemble a PIDD/ mice exposed to UV irradiation showed an increase PIP-domain located in the ﬁrst Zu5 domain in position aa 391 of apoptotic cells as compared with wild-type cells. and that is conserved in mice: ‘xAxQxxVxxW’. We mutated In summary, our results demonstrate an important role of PIDD the core sequence (QxxV converted in AxxA) and tested the in the regulation of PCNA during the DNA damage response interaction of PCNA with this mutant. Surprisingly, the induced by UV irradiation, controlling TLS and apoptosis. mutation of this sequence mostly affects the processing of PIDD, shown by the accumulation of the full-length isoform Materials and Methods (Supplementary Figure S7, lane 3 versus 2 in total extract). Cell culture and UV irradiation. Human embryonic kidney 293T cells, and For cells, it may often be preferable to risk the introduction human HeLa cervical carcinoma cells were grown in DMEM þ Glutamax (Life of point mutations during TLS over other mechanisms of DNA Technologies, Basel, Switzerland), supplemented with 10% fetal calf serum and repair, which can cause chromosomal aberrations or wide- 100 U/ml penicillin, 100 mg/ml streptomycin. The generation of stably expressing spread cell death. In cell lines lacking expression of PIDD and PIDD cells was achieved by retroviral infection as described previously, and cells thus functional TLS, we indeed found increased cell death in stably knocked down for PIDD were obtained by lentiviral infection using shRNA from Openbiosystems (Hunstville, TX, USA) as described elsewhere.29,38 UV-C accordance with published data.27 This cell death is char- irradiation were performed with a Stratalinker UV crosslinker 2400 (Stratagene, acterized by caspase-3 activation, but the nature of the Agilent Technologies, Basel, Switzerland) equipped with 254 nm tubes. Medium upstream caspase(s) leading to caspase-3 processing is was removed from cells before irradiation and replenished after irradiation. currently unclear. Caspase-2 was shown to be involved in DNA damage-induced cell death. As cell death can occur in Transient transfection. Transient transfection of both expression vectors or the absence of PIDD, the PIDD-RAIDD-caspase-2 and the siRNA were performed using the DNA-calcium phosphate precipitation method and

Cell Death and Differentiation Regulation of TLS by PIDD E Logette et al 1044

incubated for 48 h before treatment and harvesting of the cells. The total amount of antibody (AF835, R&D Systems) overnight at 4 1C at 0.5 ng/ml and revealed with an DNA was kept constant by adding empty vector where necessary. Alexa488-coupled goat anti-rabbit antibody (A11034, Life Technologies) used at 10 mg/ml in PBS 0.2% Triton X-100, then counterstained with DAPI for nuclear Coimmunoprecipitation and western blotting. Immunoprecipitation staining (Roche Diagnostics), as described in the manufacturer’s instructions. The (IP) or coIP experiments with transfected proteins were performed in lysis buffer slides were mounted in FluorSave Reagent (Merck, Nottingham, UK) and images containing 1% NP-40, 20 mM Tris, pH 7.4, 250 mM NaCl, 1 mM EDTA, 5% glycerol were acquired using a fluorescent microscope (Zeiss Axiophot, Zeiss AG, Feldbach, and a protease inhibitor cocktail. After lysis, the extracts were incubated with anti- Switzerland). Flag or anti-VSV antibody for 4 h to overnight. After incubation the beads were washed three times with lysis buffer, and analyzed by SDS-PAGE and Identification of PIDD-binding partners by mass-spectrometry immunoblotting. Cytoplasmic and nuclear fractionations. Around 106 293T cells stably For PCNA analysis, non extractable (NE) fractions were obtained with Triton expressing Flag-PIDD or control cells (obtained by retroviral infection as described X-100. Briefly, cells were lysed 1 min on ice in PBS Triton X-100 1% before in experimental procedures) were harvested and washed two times with PBS and centrifugation (10 min, 10 000 g). Supernatants were removed and pellets then two times with PBS 10 mM sodium butyrate. Cells were lysed in 2 ml lysis resuspended in sample buffer and boiled before western blotting. buffer containing 0.1% Triton X-100, 250 mM sucrose, 10 mM Tris pH 7.4, 10 mM For Cytochrome c release analysis, cytoplasmic extraction was performed with sodium butyrate, 4 mM MgCl2 and complete protease inhibitor (PI, Roche). Cells Digitonine (Sigma-Aldrich, Buchs, Switzerland). Briefly, cells were lysed in were gently disrupted in a loose Dounce homogenizer (25 strokes) and the resulting Digitonine lysis buffer (75 mM NaCl, 1 mM NaH2PO4, 8 mM Na2HPO4, 250 mM lysate was centrifugated at 4500 r.p.m. at 4 1C, 10 min. The supernatant sucrose, 100 mg/ml Digitonine) and incubated 10 min on ice before centrifugation. corresponding to the cytoplasmic fraction was cleared by centrifugation at The antibodies used for western blotting were: anti-VSV and anti-Flag 13 000 r.p.m. at 4 1C, 10 min. The pellet was resuspended in 4 ml of buffer C (Sigma-Aldrich), anti-p21 (C-19) and anti-PCNA (PC10) (Santa Cruz, Nuningen, containing 250 mM sucrose, 10 mM Tris pH 7.4, 10 mM sodium butyrate, 4 mM Switzerland), anti-mouse PIDD (Lise-1) (Adipogen, Epalinges, Switzerland), anti- MgCl2 and PI, put on a 30% sucrose cushion (30% sucrose in buffer C) and PIDD (Anto-1) and anti-Cytochrome c (Axxora, Lausen, Switzerland), anti-RFC5 centrifuged at 3300 r.p.m., 5 min at 4 1C (swing-out rotor, brake set to minimum). and anti-phospho-Chk1 (S317) (Bethyl, Lucerne, Switzerland), anti-DNA polymer- The pellet containing the nuclei was resuspended in 1 ml buffer N containing 0.5% ase eta and anti-lamin B1 (Abcam, Cambridge, UK), anti-caspase-3 (BD, Allschwil, NP-40, 10% glycerol, 420 mM NaCl, 20 mM Hepes pH 7.9, 1.5 mM MgCl2,1mM Switzerland) and anti-Bid (R&D sytems, Abingdon, UK). DTT and PI. Nuclear extracts were incubated on a rotating wheel at 4 1C for 30 min, homogenized in a loose Dounce homogenizer (20 strokes) and clarified by Chromatin extraction. For chromatin fractionation experiments, cells were centrifugation at 13 000 r.p.m. 4 1C for 20 min. Nuclear fractions were diluted before lysed 5 min on ice in solution A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, immunoprecipitations with buffer C without salt to obtain a 150 mM NaCl 0.34 M sucrose, 10% glycerol, 1 mM DTT, 10 mM NaF, 1 mM Na2VO3, proteases concentration. Tris pH 7.4 and NaCl were added to cytoplasmic fractions to inhibitors þ 0.1% Triton X-100 added just before use). Nuclei were separated by obtain a 50 mM and 150 mM concentration, respectively, before centrifugation (4 min, 1300 g), washed once in solution A (without Triton) and immunoprecipitations (described in Experimental procedures). then lysed 30 min on ice in solution B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, proteases inhibitors). Insoluble chromatin was then separated by centrifugation Fractionation, digestion and analysis by mass spectro- (5 min, 1700 g), washed once in solution B, collected by centrifugation (1 min, metry. Eluted proteins from IP were separated on a 10% mini SDS- 10 000 g), resuspended in sample buffer and sonicated before immunoblotting. polyacrylamide gel and stained with Coomassie blue. Entire gel lanes were excised into 20 equal regions from top to bottom and digested with trypsin Cytoplasm/nucleus fractionation. Cytoplasm/nucleus fractionation (Promega, Du¨bendorf, Switzerland) as described (1,2). Data-dependent LC-MS/MS experiments are based on a previous report.39 Briefly, cells were harvested and analysis of extracted peptide mixtures after digestion was carried out on a hybrid washed once in ice-cold PBS. Pellets were resuspended in 2 volume of buffer C linear trap LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Huntsville, (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 300 mM sucrose, 0.5% NP40, TX, USA) interfaced to an Agilent 1100 nanocapillary HPLC equipped with a 75 mm proteases inhibitors þ 0.5 mM DTT added at the last minute) and left 5 min on ice. internal diameter C18 reversed-phase column. Collections of tandem mass spectra Nuclei and cytosolic fractions were separated by centrifugation (45 s, 9000 r.p.m., for database searching were generated from raw data and searched using Mascot 4 1C). Nuclei were washed once with buffer C, resuspended in 2 volume of buffer (Matrix Science, London, UK; version 2.1.0) against the release 11.0 of the N (20 mM HEPES pH 7.9, 100 mM NaCl, 0.2 mM EDTA, 20% glycerol, 100 mM KCl, UNIPROT database, (SWISSPROT þ TrEMBL, http://www.expasy.org) restricted proteases inhibitors þ 0.5 mM DTT added just before use) and then subjected to to human taxonomy. The software Scaffold (version Scaffold-01_06_03, Proteome three cycles of freeze (liquid N2) and thaw (37 1C) before sonication. Nuclear Software Inc., Portland, OR, USA) was used to validate MS/MS based peptide fractions were then isolated by centrifugation (10 min, 13 000 r.p.m.). In western- (minimum 95% probability (4)) and protein (min 99% probability (3)) identifications, blots, caspase-3 and lamin-B1 were used as controls of fraction purity. perform dataset alignment and subtraction as well as parsimony analysis to discriminate homologous hits. In an additional filtering step, only proteins identified Apoptosis quantification. Apoptosis was determined by Hoechst 33342 with at least three spectra were retained for further analysis. (1 mg/ml, Sigma-Aldrich) staining of the cells for 30 min at 37 1C, and fluorescence microscopy analysis of 200 cells per condition. Conflict of interest Primary keratinocytes extraction. Adult mice (8–12 weeks old) were The authors declare no conflict of interest. killed and the tails removed. The tail skin was peeled off and cut into 1 cm fragments, which were incubated overnight at 4 1C in Dipase II solution (8 mg/ml in PBS, Roche, Basel, switzerland). The following day, the epidermis was separated, Acknowledgements. We thank N Aebi, S Roques and A Yazdi for technical incubated for 5 min at 37 1C in Trypsin/EDTA 0,05% (Invitrogen, Basel, support, all the members of the A Constantinou’s Lab and P Schneider for helpful Switzerland), and filtered through a 70 mm cell strainer. Cells were seeded on discussions, and Catherine Dostert for critical reading of the manuscript. This work collagen IV-coated plates (BD) at the density of 200 000 cells/ml, in Epilife medium was supported by grants from Hermione, Apo-SYS and ApopTrain European (Biocoba, Reinach, Switzerland) supplemented with HGKS (Biocoba) for 4 days Projects EL and MJE are supported by FEBS fellowships. before UV irradiation.

Immunohistochemistry. The irradiated skin was processed by paraffin Author contributions embedding and sections were stained by hematoxylin-eosin (HE), or using an anti- EL, SMS, AV, OG and JT designed the research, analysed the data and wrote the active caspase-3 antibody. Briefly, after hydration, paraffin sections were heated paper; EL, SMS, MJE, LXH, BJ, CM, AT and MQ performed the research. three times for 5 min in Citrate 0.01 M buffered at pH 6.0 using a microwave at 620W and cooled on ice. PBS containing 0.2% Triton X-100 and 10% normal goat serum 1. Moldovan GL, Pfander B, Jentsch S. PCNA, the maestro of the replication fork. Cell 2007; was used for blocking. The sections were then stained using anti-active caspase-3 129: 665–679.

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Cell Death and Differentiation